The pioneering work on RNA sequence determination by Dr. Holley and his group was done on transfer RNA because of its small size (77 nucleotides). The first complete analysis of a newly synthesized RNA was done on a tRNA precursor of 132 nucleotides. We are attempting to carry this process one step further, to study the mechanism of the synthesis of the tRNA precursors in a eukaryotic system with the hope that this will be more readily elucidated than the synthesis of mRNAs in eukaryotes because of the smaller size of tRNAs compared with mRNAs. We will proceed with our work on the DNA-dependent RNA polymerase III of yeast which is a good candidate for the enzyme responsible for the synthesis of tRNA. The enzyme as isolated, as well as I and II from yeast and other eukaryotes, does not include a sigma- type factor. To get the correct initiation and termination involved in synthesis of tRNA precursors, we will have to determine if indeed there is a sigma-type factor and a rho-type factor involved in eukaryotic transcription. We will also continue our work on the modification of nucleotides in tRNAs by working out the mechanism of the conversion of uridines in one of the loops of tRNA into dihydrouridines.